2013~2017年江西省猪圆环病毒2型分子流行病学调查

为掌握江西地区猪圆环病毒2型（PCV2）的分子流行病学及其遗传变异情况,本研究运用实验室已建立的PCR方法对2013~2017年采集自江西省南昌市、宜春市、新余市、赣州市、吉安市、九江市、上饶市、抚州市、景德镇市、鹰潭市等10个地区的1 082份疑似PCV2感染猪病料组织进行病原学检测,并通过PCR扩增、克隆和基因测序对PCV2的全基因组序列进行分析。研究表明,1 082份病料中有610份为PCV2阳性,总阳性率为56.4%,其中2013~2017年阳性率分别为52.7%、48.8%、58.5%、71.0%和67.4%。共获得89株PCV2全基因组序列,其中有83株为PCV2b基因型,其中53株归类于PCV2b-1C基因亚群,30株归为PCV2b-1A/1B基因亚群;6株为PCV2a型。测序毒株与参考毒株ORF2基因核苷酸、氨基酸序列同源性分别为88.9%~100.0%和86.0%~100.0%;氨基酸序列分析表明,Cap蛋白存在20个氨基酸突变位点。本研究表明,江西地区猪群中PCV2的主导基因型为PCV2b,其中又以PCV2b-1C基因亚群占据主导地位,同时也存在少量PCV2a基因型。     

猪圆环病毒2型毒株分离鉴定和体外增殖特性研究

为分离鉴定猪圆环病毒2型(PCV2)毒株并研究其体外增殖特性,本研究采用PCR法分别对从山东、山西、北京和河北收集的疑似断奶仔猪多系统衰竭综合征(PMWS)病猪的病料进行检测,将检测结果为PCV2阳性的病料处理后接种PCV阴性的PK15A细胞连续传代进行病毒分离,观察每代细胞是否有病变,采用PCR、间接免疫荧光法(IFA)对第3代培养物进行鉴定,对各毒株的全基因组进行克隆、测序和系统进化分析,并将PCV2各分离株连续传20代,对其体外增殖特性进行了研究。结果共分离到5株猪圆环病毒2型毒株,分别命名为DBN-SD02、DBN-SX07 DBN-BJ08、DBN-HB12和DBN-HB13株,GenBank登录号分别为FJ660967、FJ660968、FJ660969、FJ660970和FJ660971。各分离株基因组全长均为1767 bp,系统进化分析结果表明,除DBN BJ-08属于PCV2a基因型外,其余均属于PCV2b基因型,基因组序列核苷酸同源性为98.5%~99.7%,体外增殖特性研究结果表明,随着传代次数的增加病毒感染滴度均有明显的提高。     

中国部分地区猪圆环病毒2型的基因型分析

对中国11个省(市)部分猪场收集的812份病料,应用ORF2-PCR进行检测,发现有235份为PCV2阳性病料.利用鉴别PCR方法从235份阳性病料中,检出10份PCV2a基因型和133份PCV2b基因型,检出率分别为4.2%、56.6%.进一步对ORF2-PCR检测阳性的部分模板进行全基因组扩增,构建阳性重组质粒,对插入质粒的片段进行测序鉴定,利用DNAStar进行同源性分析,同GenBank参考毒株绘制系统发育树,从系统发育树中也可分出PCV2a、PCV2b 2种基因型.鉴别PCR和测序结果说明,我国部分地区流行的猪圆环病毒2型以PCV2b基因型为主,少数为PCV2a基因型,也有既非PCV2a又非PCV2b的基因型(PCV2c?).     

猪圆环病毒2型分离株优势基因型分析与检测

为探讨猪圆环病毒2型(PCV2)流行株基因型的变化,本研究对1999年～2009年NCBI中登录的556个PCV2分离株全基因序列进行分析。结果表明,1999年～2002年所登录的PCV2的优势流行毒株为基因A型(PCV2A);从2003年开始基因B型(PCV2B)逐渐为优势流行毒株,在PCV2分离株中基因B型所占比例,2004年为95.4%,2007年～2009年均超过76.0%。2003年～2009年国内的PCV2分离株有287个,其中250个为PCV2B,37个为PCV2A,表明PCV2B是国内猪群中的主要优势毒株。通过建立PCV2A和PCV2B PCR检测方法,对70份临床病料的检测显示,PCV2检出率为34.29%(24/70),基因型均为PCV2B,无PCV2A的检出,表明PCV2B为检测猪群中的主要优势毒株。     

猪圆环病毒2型广东分离株的遗传演化分析

为了解广东地区猪圆环病毒2型(PCV2)毒株的遗传变异情况,从广东不同地区的规模化猪场采集疑似断奶仔猪多系统衰竭综合征(PMWS)病猪的淋巴结、肺脏和脾脏。对PCR鉴定为阳性的病料在PK-15细胞上增殖培养6代,采用PCR和相关分子生物学软件对12个分离株进行全基因组克隆和测序分析。结果显示,分离株基因组全长为1 767 nt或1 768 nt,核苷酸序列同源性为94.6%~99.9%。遗传演化分析表明,12个分离株分布于3个大群,其中5株属于PCV2b,4株属于PCV2d,3株属于PCV2e,提示广东地区PCV2流行毒株已发生变化。     

猪圆环病毒2型河南株全基因组的克隆与序列分析

利用PCR方法,对河南郑州、南阳、焦作等地采集的疑似猪圆环病毒2型（PCV2）感染的病料进行检测。PCR检测为阳性的病料经处理后,接种无PCV污染的PK-15细胞中盲传6代,克隆11株PCV2全基因组并进行序列分析。结果表明,11株PCV2中有10株基因组全长为1 767bp,基因组分型为PCV2b,1株为1 768bp,说明PCV2b是河南省断奶后多系统衰竭综合征（PMWS）发生的主要因素;11个毒株的同源性位于94.9%～100%,与其他毒株的同源性为94.7%～100%;10株基因组为1 767bp的毒株处于一大分支上,遗传进化比较稳定;本试验为PCV2在河南地区的分子流行病学、遗传变异及防治奠定了基础。     

猪圆环病毒2型北京株的分离及基因组序列分析

为了解北京地区猪圆环病毒2型(PCV2)毒株的遗传变异特性,本研究对2010年病料中分离的2株PCV2通过PCR、IPMA进行初步鉴定,并扩增病毒全基因组,用DNAStar进行序列分析,用MEGA5进行PCV2ORF2序列比对,构建系统进化树。结果表明,分离得到的BJ2010LC株(登录号为JQ002671)、BJ2010PG株(登录号为JQ002672)2个PCV2毒株其全基因组长度均为1 767bp,与国内外参考毒株核苷酸序列同源性为94.2%～99.5%,2个分离株之间的核苷酸序列同源性为96.6%。BJ2010LC株属于基因型PCV2d,BJ2010PG株属于基因型PCV2b。     

猪圆环病毒2型2015JS株的分离鉴定及全基因序列分析

采用细胞传代法对PCR检测为猪圆环病毒2型(PCV2)阳性的病料进行病毒分离,通过PCR、间接免疫荧光试验(IFA)对分离的病毒进行初步鉴定,应用IFA测定分离株的TCID50。应用PCR特异性扩增出该分离株的全基因组,经测序后进行同源性和系统进化分析。结果:成功分离到1株PCV2毒株,全基因组长度为1 766 bp,命名为2015JS株,分离株在细胞上的TICD50为10-5.50,与乌拉圭毒株Uy99(Gen Bank登录号KP867050)的同源性最高(99.8%),同属PCV2d型;与丹麦PCV2c毒株DK1980PMWSfree(Gen Bank登录号EU148503)的同源性最低(94.3%)。研究结果为江苏PCV2流行病学的研究和遗传变异的防控提供了参考资料,也为江苏PCV2疫苗毒株提供了选择依据。     

猪圆环病毒2a/2b亚型病毒株毒力分析

本研究对367份猪内脏样本进行PCV2检测,对阳性样本进行2a/2b鉴别诊断,并采用ORF2测序方法对部分阳性样本进行测序。在发病猪群中PCV2b阳性率远高于健康猪群,而健康猪群中PCV2a的阳性率高于发病猪群,从而在临床表型方面论证了PCV2b毒力强于PCV2a。通过比较12株PCV2 ORF2推导氨基酸序列的关键氨基酸位点,9株PCV2b毒株在ORF2氨基酸76位为I,131位为T;而3株PCV2a毒株在ORF2氨基酸76位为L,131位为P,据比对结果可知,PCV2b毒株为强毒株,PCV2a毒株为弱毒株。研究结果表明,PCV2的2种亚型2a/2b无论是临床表型还是ORF2基因型,毒力表现都是有差异的,且PCV2b毒力要强于PCV2a。     

2010-2013年四川地区猪圆环病毒2型的分子流行病学调查

为了掌握四川地区近年猪圆环病毒(PCV2)的分子流行和分子进化动态规律。将2010-2013年期间采集自四川成都、德阳、雅安、西昌、遂宁、乐山、资阳等11个养猪地区的疑似PCV2病料共324份进行了检测与分析。设计1对检测PCV2的特异性引物,PCR检测病料表明PCV2阳性为229份,PCV2感染率为70.7%。随后对本实验室分离到的11株PCV2分离毒株进行全基因组克隆与序列分析,11株PCV2四川分离株的全基因组序列分析均为PCV2b基因型,各地分离株之间的全基因同源性为95.2%⁹9.7%,与参考毒株之间的全基因同源性分别为94.1%99.7%,与参考毒株之间的全基因同源性分别为94.1%⁹9.7%;与国内外参考毒株相比,ORF1的核苷酸及推导氨基酸的序列的同源性分别为96.7%99.7%;与国内外参考毒株相比,ORF1的核苷酸及推导氨基酸的序列的同源性分别为96.7%¹00%、98.4%100%、98.4%¹00%,ORF2核苷酸及推导氨基酸的序列的同源性88.0%100%,ORF2核苷酸及推导氨基酸的序列的同源性88.0%⁹9.7%、85.4%99.7%、85.4%¹00%,序列分析发现ORF2变异位点较多,而ORF1相对较保守。本研究结果显示:2010-2013年间,四川地区PCV2感染率高,主要分布在乐山、德阳等市,且流行的基因型以PCV2b为主。     

2008～2011年中国部分地区猪圆环病毒2型的分子流行病学调查

为了解2008~2011年中国部分地区猪圆环病毒2型(Porcine circovirus type 2,PCV2)分子流行病学变化趋势,本实验室共采集福建省、江西省、广东省、安徽省、浙江省、河南省、河北省、广西壮族自治区、内蒙古自治区、上海市、江苏省和山西省共12个省(市、区)的健康猪群和发病猪群共452份样品,对其进行病原学检测,并通过扩增、克隆和测序共获得31株PCV2 ORF2基因编码序列。结果显示,452份样品中,有354份样品检测为PCV2阳性,感染率高达78.3%。对31株ORF2基因序列的分析和比对结果表明,31株PCV2均为PCV2b基因型,其中有21株归类于PCV1A/1B基因亚群,而10株为PCV1C亚群;对31株PCV2 ORF2编码氨基酸序列比对分析表明PCV2基因亚型具有其特异的氨基酸变异位点,这对于临床上区别PCV2亚型具有一定的指导意义。从基因水平上来说,自2008年以来中国以PCV 1A/1B亚群为主要流行致病株,但值得我们关注的是,PCV 1C亚群毒株从无到有并有逐渐增多的趋势,将来可能在PCV2的流行毒株中占据主要地位。     

Detection of porcine Circovirus type 2 (PCV2) variants PCV2-1 and PCV2-2 in Brazilian pig population

In the present study whole genome of six Brazilian isolates of PCV2 were sequenced, analyzed and compared with 35 other sequences (24 from other countries and 17 from Brazil). The phylogenetic analysis showed that mostly Brazilian variants of PCV2 were grouped as PCV2-1. Two isolates among the six analyzed here could not be grouped with any other PCV2-2 analyzed in this study. One of these isolates was from an aborted fetus with myocarditis and the other from a PMWS affected pig. The results pointed here showed that both groups of PCV2 are present in Brazilian pig population without any clear geographical correlation.     

Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case-control study supports a shift in genotypes with time

Porcine circovirus type 2 (PCV2) is the primary cause of Postweaning Multisystemic Wasting Syndrome (PMWS) in pigs. PCV2, however, is found in both PMWS-affected herds and non-affected herds. The objective of this study was to clarify if PCV2 genome nucleotide sequences isolated from pigs from PMWS-affected herds and non-affected herds cluster phylogenetically in two separate groups. All isolates (45) belonged to PCV2 group 1 and shared a nucleotide sequence identity of 99.4-100% indicating a very homogeneous PCV2 population in Denmark. Phylogenetic analysis of the PCV2 isolates revealed no distinctive clustering of case- and control-herds suggesting that there is no link between PCV2 sequences and herd disease status. The appearance of only PCV2 group 1 isolates in this study (isolates from 2003/2004) led us to determine if PCV2 nucleotide sequences had changed in Denmark over time. Interestingly, all PCV2 isolates from before the first outbreak of PMWS (2001) belonged either to a new PCV2 group identified for the first time in this study and named group 3 (isolates from 1980, 1987 and 1990) or PCV2 group 2 (isolates from 1993 and 1996). The shift from PCV2 group 2 to 1 was confirmed on a more global scale by placing all full genome PCV2 sequences submitted to GenBank from 1997 to 2006 in either of the groups by phylogenetic analysis. The analysis showed that the shift happened in 2003 or even earlier. This may indicate that PCV2 group 1 is a more adapted form of PCV2 and possibly could be more pathogenic.     

First description of postweaning multisystemic wasting syndrome (PMWS) in wild boar (Sus scrofa) in Croatia and phylogenetic analysis of partial PCV2 sequences

This report describes the first case of postweaning multisystemic wasting syndrome (PMWS) in wild boar in Croatia. During the winter season of 2004, eight wild young piglets (of approximately 2 to 5 months of age) were found dead in a fenced hunting area. Polymerase chain reaction (PCR) was carried out on mesenteric lymph nodes and all animals yielded positive results. In one of these animals diagnosis of PMWS was established based on the three key diagnostic criteria including the clinical manifestation, moderate lymphoid lesions consisting of lymphocyte depletion and granulomatous inflammation, and detection of the presence of PCV2 genome within the lymphoid lesions by in situ hybridisation (ISH). Three additional wild piglets had also mild PMWS-like lesions and a low amount of PCV2 was also found. No PMWS-like lesions or PCV2 genome were detected in the rest of the wild piglets studied. Three PCR-positive isolates were partially sequenced, which confirmed the diagnosis of PCV2 and demonstrated that the three sequences were genetically identical. The phylogenetic analysis of a representative PCV2 isolate indicated that its sequence (DQ875444) is grouped in a separate branch with Hungarian isolate (AY256460) and differs from any of the annotated sequences.     

PCR detection and characterization of type-2 porcine circovirus.

A polymerase chain reaction (PCR) assay was developed for detecting porcine circovirus (PCV). The assay readily detected type-2 PCV (PCV-2) and type-1 PCV (PCV-1). The PCR primers were designed based on DNA sequences conserved in all reported PCV genomes. Type 1 PCV and type 2 PCV both produced 438 bp amplification products, which were easily identified and differentiated from one another by restriction fragment length polymorphism (RFLP) analysis. Porcine circovirus was detected in 55% (931/1693) of randomly tested pigs with various clinical signs and lesions, most of which were difficult to differentiate from those associated with porcine reproductive and respiratory syndrome (PRRS). The PCR products from all positive clinical samples were identified by RFLP to be only PCV-2; DNA tested by PCR was extracted directly from one or more of lung, mesenteric or mediastinal lymph nodes, and tonsil. Type 2 PCV was also detected in 6% (2/34) of DNA extracted directly from semen of randomly chosen healthy boars. Positive PCR reactions from 554 diseased pigs were characterized by RFLP and categorized into 5 different profiles (A-E), of which 82.8% were PCV-2A (456/554), 3.0% were PCV-2B (17/554), 9.9% were PCV-2C (55/554), 1.1% were PCV-2D (6/554), and 3.2% were PCV-2E (18/554). The complete genomic nucleotide sequences of PCV-2A, B, C, D, and E were determined and found to have at least 95% homology compared with one another and with all other PCV-2 found in the GenBank database. All PCV-2 had less than 76% homology with PCV-1. This PCR assay will hopefully be useful to veterinary diagnostic laboratories for routine testing and surveillance of infection with PCV-2. The RFLP profiling system might be useful for preliminary characterization and identification of PCV isolates and might also benefit studies on the molecular epidemiology of PCV.     

猪圆环病毒2型江苏分离株的遗传进化分析

采用PCR方法扩增了15个猪圆环病毒2型（PCV 2）江苏分离株的基因组DNA,以这些毒株的ORF2核苷酸序列进行遗传进化分析。经序列比较发现,所有分离株均属于2b基因群,其中7株为1A/1B亚群、8株为1C亚群;毒株间核苷酸同源性为93.6%~100%,所编码的Cap蛋白氨基酸同源性为92.3%~100%。PCV 2江苏分离株Cap蛋白的主要变异区域为53~90、121~151和190~210位氨基酸;R59、R89、S90、S121、T134、S169、A190、E210为1A/1B亚群分离株的特征氨基酸,而F8、I53、N68、L89、T90、T121、N134、R169、D210、I215、K234则是1C亚群分离株的特征氨基酸。     

Characterization of porcine circovirus type 2 in Taiwan

In an effort to understand the genetic diversity of porcine circovirus type 2 (PCV2) and the prevalence of PCV2 infection in Taiwanese herds, we have sequenced the complete genomes from PCV2-infected specimens and individually measured the antibody titer against PCV2 from pigs reared in Taiwan between the years 2000 and 2002. A total of 623 specimens originating from pigs displaying varied clinical signs were screened with the polymerase chain reaction (PCR). Results showed that 309 pigs (49.6%) tested positive for PCV2. Eight of the positive specimens were used for the amplification of the complete viral genome. Sequence comparison of the complete genomes indicated that the 8 Taiwanese PCV2 isolates shared 95-99% similarity. Phylogenetic analysis of all 40 PCV2 isolates from North America, Europe, Asia and Taiwan revealed that those isolates were grouped together in one large group containing two minor subgroups. The Taiwanese PCV2 isolates were classified into the two minor subgroups. The prevalence of serum antibodies to PCV2 in pigs was investigated, and results showed that approximately 83.5% of the pigs in Taiwan were seropositive. Finishing pigs possess the highest titers of antibodies, while 9-week-old pigs contained the lowest titers for specific antibodies. Our results suggest that PCV2 infections have become common in Taiwanese pig farms.